1. Field of the Invention
The invention relates to a point-scanning luminescent microscope, especially for studying biological objects, with at least one collimated light source for producing an excitation light beam, an optical arrangement which focuses the light of the excitation light source onto the object to be examined, at least one detector arrangement for acquiring the light emitted by the object, an optical arrangement which collects the light emitted by the object and sends it to the detector arrangement, and a scanner arrangement which causes relative movement between the scanning light beam and the object in at least two directions.
2. Description of Related Art
FIG. 1 shows a schematic of a luminescent microscope of the type to which the present invention is directed and which is also called a scanning microscope. The important assemblies of the luminescent microscope shown here are:
a collimated light source 10, generally a laser, for excitation of the object 12; PA1 an optical arrangement which is designated 14 as a whole and which focuses the light of the excitation source 10 as an illumination spot, especially as a diffraction-limited spot, onto the object 12, and which, at the same time, collects the emitted light again and sends it to a detector 16 or 18 (see below), PA1 a scanning device moves either the illumination spot relative to the object to be examined (scanning device 20) or the object to be examined relative to the spot (scanning device 22) and thus is used for scanning, and PA1 one or more detectors 16 or 18 for recording the light emitted from the object 12.
Such a point-scanning luminescent microscope can be designed especially as a confocal microscope and/or as a microscope which operates using the twin-photon or multi-photon process.
In the first case, the detector(s) is(are) positioned behind a confocal diaphragm 24 (detector position 16) which is attached in the image plane at a point conjugate to the illuminated object point. As FIG. 1 shows, here, the beam path of the light emitted by the object 12 to be examined runs via the optical arrangement 14, which is used to focus the excitation light onto the object, and via a dichroitic beam splitter 26, which separates the emission light from the excitation light, and a lens 28 to the confocal diaphragm 24 and the detector located in the detector position 16. In this way, light from other planes is largely masked as the focal plane of interest and three-dimensional scanning becomes possible.
In one special form of the scanning luminescent microscope indicated above, the sample is excited by a nonlinear twin-photon or multi-photon process which per se is limited to the focal plane and this, in most cases, makes a confocal diaphragm unnecessary. In this case, it is not necessary to allow the emitted light to pass through the entire optical arrangement backwards. A detector can also be placed directly behind the objective lens (no "descanning"). As indicated in FIG. 1, for this reason, in the beam path of the microscope 14, i.e. between the objective lens 32 and the tube lens 34, there can be a dichroitic beam splitter 30 so that the light emitted by the object is decoupled from the beam path directly after passing through the objective lens 32 and is imaged via another tube lens 36 onto the detector located in the detector position 18. The light collected at a given time always originates from the focal point which has just been illuminated, as dictated by the principle.
Although promising results have been obtained with a luminescent microscope built in this way, there a need for further improvements in this area.